Precolicin E1, the major gene product of plasmid-ColE1 deoxyribonucleic acid in vitro

Coupled transcription and translation of plasmid-ColE1 DNA in vitro under optimized conditions gave one major product. This has an apparent weight of 71 000, the same N-terminal sequence as colicin E1 and was not digested by deoxyribonuclease or ribonuclease. It differed from colicin E1 in its C-terminal residue and amino acid composition. It had lower specific activities in cell killing and in the fluorescence-enhancement in vitro assay of Phillips & Cramer [(1973) Biochemistry 12, 1170--1176] than did colicin E1, but both proteins bound in equimolar amounts to colicin-sensitive and colicin-resistant cells. The product of plasmid-ColE1-DNA-directed protein synthesis was converted into a protein indistinguishable in structure and activity from colicin E1 by incubation in the reaction mixture, after deoxyribonuclease and ribonuclease treatment, for a further 20 h at 37 degrees C. A protein with similar properties to the 71 000-dalton product in vitro was identified in extracts of a ColE1+ colicin-tolerant mutant of Escherichia coli K12. It is concluded that this protein probably represents a pre-form of colicin E1 which may be involved in colicin-E1 secretion or cellular colicin-E1 immunity in colicin-E-producing cells, or both of these processes.